Screening and identification of novel specific markers of breast cancer stem cells.

Abstract

Breast cancer is the leading cause of death among women worldwide. Until recent years, triple negative breast cancer could be divided into 6 types according to different biomarkers with the development of sequence and microarray technology. However, these results rarely have therapeutic impact and still lack validation with the string criteria of clinical studies. Therefore, the present study aimed to screen novel markers of breast cancer stem cells and to verify the specificity in vitro and in vivo. In the present study, screening for phages specifically binding to breast cancer stem cells was performed, positive phage DNAs were extracted, and polypeptides were synthesized and labeled with FITC. The specificity of the polypeptides was identified in vitro and in vivo. Breast cancer stem cells were cultured and identified by flow cytometry. A phage random-peptide library was amplified and screened by culturing with breast cancer cells and breast cancer stem cells. The positive phage was identified by ELISA, and positive phage DNA was extracted. The DNA pellet was isolated and sent for external sequencing with the primer −96 gIII. Based on the sequencing results, a polypeptide was synthesized and labeled with FITC. The specificity to breast cancer stem cells was identified in vivo and vitro. Following three rounds of screening, the phage was enriched ~200-fold. Immunofluorescence demonstrated that two randomly selected phage clones, B8 and A3, had specific affinity to breast cancer stem cells. The results of the present study indicated that phage polypeptides that specifically bind to breast cancer stem cells were successfully screened through stem cell enrichment and phage display technology, which may be beneficial for targeted therapy and further study of breast cancer stem cells.